The research program outlined in this proposal concerns the study of mechanisms of enzymic and non-enzymic phosphoryl transfer reactions. Procedures for the determination of heavy atom isotope effects on phosphoryl transfer reactions using the isotope ratio method will be developed. These techniques along with others will be used in an in-depth study of the catalytic mechanism of two unique phosphoryl transferring enzymes. The first of these, yeast inorganic pyrophosphatase, catalyzes the transfer of a phosphoryl group from pyrophosphate to water and thus represents a simple model to enzymes catalyzing more complex chemical transformation The second enzyme catalyzes the rearrangement of phosphoenol pyruvate to pyruvate phosphonate in Tetrahymena pyriformis. The importance of this reaction derives from its key position in the biosynthetic pathway of phosphonolipids and from the novelty of the chemical transformation involved. Specifically, a carbon atom rather than oxygen atom serves as the phosphoryl acceptor and hence a phosphonate, a species rarely observed in nature, is formed rather than a phosphate. The Tetrahymena enzyme, yet unnamed, will be purified and subjected to a detailed mechanistic study.